Antiviral compositions and method of administration



United States Patent 3,274,054 ANTIVIRAL COMPOSITIONS AND METHOD OFADMINISTRATION Andrew Stephen Tomcufcik, Tappan, Robert Bruce Angier,Pearl River, Martin Forbes, New City, and Howard Frederick Lindh,Monsey, N.Y., assignors to American Cyauamid Company, Stamford, Conn., acorporation of Maine No Drawing. Filed June 28, 1963, Ser. No. 291,28211 Claims. (Cl. 167-65) This invention relates to methods of and newcompositions for treating viral infections in warm blooded animals.

Heretofore no chemotherapeutic agents have been found which have provedeffective in treating systemic virus infections. We have now found thatcompositions containing as an active ingredient a compound of thefollowing formula are useful in treating viral infections in warmblooded animals:

R is hydrogen or lower alkyl, R and R are hydrogen or lower alkyl, R andR are lower alkyl, X is lower alkylene, Z is oxygen or sulfur and acidaddition and quaternary salts thereof.

The free bases of the compounds of this invention are, in general, oilyliquids or low melting solids. They are, in general, soluble in waterand in the usual organic solvents, such as lower alkanols, acetone,lower alkyl esters of lower alkanols, benzene, toluene, ethers and thelike.

The free bases of the compounds of this invention form acid additionsalts with acids such as hydrochloric, hydrobromic, sulfuric, acetic,citric, tartaric, lactic, succinic, maleic, phosphoric, malic, gluconic,ascorbic, 1,1- methylene-bis-(2-naphthol-3-carboxylic acid),butanel,2,3,4-tetracarboxylic acid and the like. Such acid additionsalts are in general soluble in water, but not soluble in non-polarorganic solvents such as ethers and petroleum ethers; and they may beprepared, in general, by adding an anhydrous acid to an anhydroussolution of the basic compound in ether or alcohol and thereafterisolating the precipitated salt by filtration.

The compounds of the present invention can be prepared by methodsdescribed hereinafter in the examples and in the prior art. The mostgeneral method for preparing the present compounds is the reaction ofl-carbethoxy-4-lower alkylpiperazine with a di-loweralkylamino lowerfatty acid alcohol. Other methods are shown hereinafter in the examples.

One of the compounds of the present invention, diethylaminoethyl4-methyl-l-piperazinecarboxylate, has shown a reproducible protectiveeffect in experimental influenza A virus infections in mice. The resultsof tests are sum marized in the following Table I.

3,274,054 Patented Sept. 20, 1966 TABLE I.-EFFECT OF Z-DIETHYLAMINOETHYL4-METHYLPIPERAZINE-l-CARBOXY LATE IN IN- FLUENZA INFECTION IN MICESingle dose treatment Survivors/Group on 14th day after Infection SingleDose, rug/kg.

Oral Subcutaneous Treatment Treatment 15 Controls (0.85% NaOl) 5 1o0 1Animals: Taconic male white mice; 16-20 grams body weight. Inigztion:Intranasal; 0.05 ml. of a 10' broth dilution of virus stock 0.Treatment: Single dose directly after infection; oral doses by tubing of0 1.0 ml. volume of aqueous solution; subcutaneous doses by injection of0.5 ml. volume of aqueous solutions; all solutions adjusted to pH 7.0.

The above Table I summarizes the results with a single dose as treatmentdirectly after infection. The following Table II summarizes results withthree dose oral treatment.

TABLE II.EFFECT OF Z-DIETHYLAMINOETHYL 4 METHYLPIPERAZINE l CARBOXYLATEIN THE INFLUENZA INFECTION IN MICE Three dose oral treatment InfectionIntranasal; 0.05 ml. of a 10- broth dilution of virus stock No. 67.

Treatment: Oral tubing of 1.0 ml. volume of aqueous solutions at 0, 24and 48 hours after infection.

Other compounds of the present invention were injected subcutaneouslyand the results obtained are summarized in the following Table III.

TABLE III.EFFECT OF OTHER COMPOUNDS ON 50 SURVIVAL RATIO OF MICEINFECTED WITH INFLUENZA A (PR-8 VIRUS) Survival Name Dose, rug/kg. Ratioon 14th Day Dimethylaminoethyl 4-methylpiperazine- 800 5/15-carboxylate. 3-Dimethylarninopropyl4methy1pipera- 800 5/15zinel-carboxylate. 0 Z-Dimethylaminoisopropyl 4-methyl- 800 5/15piperazine-l-carboxylate. Controls (0.85% NaCl) 5/105 Animals: TaconieFarms male white mice; 19-25 grams body weight Infection: Intranasal;0.05 ml. of a lCldilution in brain heart infusion broth of stock PR-8virus. Treatment: Compounds dissolved in water, pH adjusted to 7.0.Indicated dose imected subcutaneously immediately after infection.Controls received 0.85% NaCl solution.

The compounds of the present invention described above Will be dispensedin compositions comprising the active ingredient and excipientsincluding a carrier. While the amount of active ingredient to be givendaily will depend on many factors such as the size, weight, age, kind,and severity of infection, etc. of the warm blooded animal, a dailyintake ranging from 5 to 500 milligrams per kilogram of body weight willproduce good results. The dosage unit may vary from 0.05 to 2 g. and may'be in a form to be administered one or more times per day, or insmaller forms for multiple daily, or other more frequent administration.Any of the usual dosage unit forms of pharmaceutical compositions can beuseful, for example, tablets, Oblets, hard or soft shell capsules,parenteral solutions or suspensions, oral solutions or syrups,

etc. I

Tablets or Oblets may include, in addition to active ingredient, any ofthe following excipients. A binder such as acacia, corn starch, gelatin,or the like. A disintegrating agent such as corn starch, potato starch,alginic acid, or the like. A lubricant such as stearic acid, magnesiumstearate, talc or the like. Also a sweetening agent such as saccharin orsucaryl, and/ or a flavoring such as peppermint oil, oil of Wintergreen,or orange or cherry flavoring can be used.

Capsules may include, in addition to active ingredient, a lubricant andalso an inert filler such as lactose, sucrose, corn starch or the like.

Solutions, usually include an acid such as hydrochloric, citric,tartaric, succinic, maleic, ascorbic, phosphoric or the like, or asuitable bufler thereof. Suspensions include a surfactant such aspolyoxyethylene sorbitan monooleate (a complex mixture ofpolyoxyethylene ethers of mixed partial oleic esters of sorbitolanhydrides); oxyethylated tertiary octylphenol formaldehyde polymer;p-isooctylpolyoxyethylenephenol polymers or the like; a suspending agentsuch as polyethylene glycol 4000 USP (a condensation polymer of ethyleneoxide and water, represented by the formula: HOCH (CH OCH ),,CH OH,wherein n varies from about 70 to 85, the molecular weight being about4000), carboxy methylcellulose (sodium carboxymethylcellulose USP, thesodium salt of carboxymethyl ether of cellulose having a closelycontrolled number of sodium carboxymethyl (CH COONa) groups introducedinto the cellulose molecule to bring about solubility in water);methylcellulose (cellulose methyl ether prepared from wood pulp orchemical cotton by treatment with alkali and methylation of the alkalicellulose with methyl chloride), and the like; and a buffer such as aphosphate, citrate, or tartrate buffer. Both solutions and suspensionsmay include a stabilizer such as disodium salt ofethylenediaminetetraacetic acid, sodium sulfite, monothioglycerol, orthe like; and a perservative such as benzyl alcohol, parabens (methyland propyl esters of phydroxybenzoic acid), or the like.

Oral solutions may include a suspending agent or viscosity control suchas magnesium aluminum silicate, carboxymethylcellulose or the like asWell as a buffer, preservative, etc. Solutions and suspensions may be ofthe aqueous sugar or sorbitol type. It is also known in pharmaceuticalpractice to employ a propyleneglycol type solvent for use with drugs.

The invention is further illustrated by means of the following examples.

Example I.Preparati0n of Z-dimelhylaminoethylpiperazine-I-carboxylate Ina flask connected to a condenser and receiver is placed 817 g. ofZ-dimethylaminoethanol, which contains 8 g. of dissolved sodium, and 474g. of l-carbethoxypiperazine. The mixture is heated first between 6070C. for 7 hours at 32-38 mm. pressure and then at 140 C. for 7 hours atatmospheric pressure. The resultant solution is carefully fractionatedat reduced pressure to remove excess 2-dimethylaminoethanol andunreacted l-carbeth- 4 oxypiperazine. A crude fraction, boiling point140-160 C. at 1525 mm. is collected and redistilled to yield 2-dimethylaminoethyl piperazine-l-carboxylate, a colorless oil, boilingpoint 114-117 C./0.51 mm., 21 1.4823.

Example lI.-Preparation of Z-diethylaminoethylpi perazine-I -carboxylate Four g. of sodium are dissolved in 928 g. of 2-diethylaminoethanoland 474 g. of l-carbethoxypiperazine are added to the solution. Themixture is then heated in a manner similar to that described in ExampleI, first at reduced pressure, in this case at 40 mm. for 7 hours at 88C., and then at the boiling point of Z-diethylaminoethanol for 7 hoursat atmospheric pressure. The Z-diethylaminoethylpiperazine-l-carboxylate is a colorless oil, boiling point l44.5-147C./4 mm., n 1.4803.

Example IlI.Preparati0n of Z-diethylaminoelhyl4-methylpiperazine-1-earb0xylate A mixture of 468 g. ofZ-diethylaminoethanol containing 4 g. of dissolved sodium and 172 g. of1-carbethoxy-4- methylpiperazine is heated under reduced pressure and atatmospheric pressure in a manner similar to that described in Example11. Purified material consists of 2-diethylaminoethyl4-methylpiperazine-l-carboxylate, a colorless oil, boiling point l27-128C./ 1 mm., 11 1.4700.

Example I V.Preparati0n of Z-diethylaminoethyl4-methylpiperazine-I-carb0xylate dihydrochloride A salt is prepared bypassing hydrogen chloride gas into an ethereal solution of2-diethylaminoethyl 4- methylpiperazine-l-carboxylate of Example III.The product, 2- diethylaminoethyl 4-methylpiperazine l-carboxylatedihydrochloride, is obtained as a white solid which melts afterrecrystallization from alcohol-ether at 196.3- 198.3 C. witheffervescence.

Example V.Preparation of 3-diethylam-in0propyl4-methylpiperazine-1-carb0xylate Metallic sodium (0.4 g.) is dissolvedin 51.5 g. (0.39 mole) of 3-diethylamino-l-propanol followed by theaddition of 17.2 g. (0.10 mole) of 1-carbethoxy-4-methylpiperazine. Thesolution is heated in an oil bath at 90 C. for seven hours under reducedpressure (25-50 mm. of Hg) and then refluxed for ten hours atatmospheric pressure. The excess aminoalcohol is removed on a steam bathat 20 mm. of mercury and the residue is distilled through a smallVigreux column. The fraction boiling at 114l23 C. (0.6 mm. of Hg) iscollected; yield 11.2 g., n =1.4702. This product is redistilled througha Nester-Faust spinning band column, yield 7.5 g., boiling point 126-l28C. (0.9 mm. of Hg), n =1.4712.

Example Vl.-Preparation of Z-dimethylaminoisopropyl4-methylpiperazine-1-carb0xylate Metallic sodium (0.4 g.) is dissolvedin 41.3 g. (0.4 mole) of l-dimethylamino-2-propanol. To this solution isadded 17.2 g. (0.1 mole) of 1-carbethoxy-4-methylpiperazine and thenheated for six hours at 80-90 C. A stream of dry air is passed throughthe reaction mixture at the beginning of the heating period. This causes15 ml. of the aminoalcohol to distill over which is added back to thereaction. At the end of the six hours several ml. of distillate arecollected before refluxing for an additional eight hours. The mixture isfiltered and the filtrate is distilled through a Nester-Faust spinningband column, yield 2.4 g., boiling point 84-88 C. (10.4 mm. of Hg), n1.4662.

Example VII.-Preparati0n 0f Z-dimethylaminoethyl 4-methylpiperazine-1-carb0xylate To 36 g. (0.4 mole) of Z-dimethylaminoethanol is added 4.6g. (0.2 mole) of sodium followed by brief refluxing to completesolution. The solution is then cooled to room temperature, diluted with50 ml. of diglyme and 22.5 g. (0.11 mole) of4-methylpiperazine-l-carbonyl chloride Example VIII.-Preparation= 09Z-diethylaminoetlzyl 4-methylpiperazine-I-carb0thi0late dihydrochloride2-diethylaminoethanethiol hydrochloride (32.8 g., 0.193 mole) isdissolved in 25 ml. of water and covered with 100 ml. of ether.Approximately 50-60 g. of anhydrous potassium carbonate is addedfollowed by vigorous shaking. The ether layer is decanted and theremaining slurry is shaken with three additional 100 ml. portions ofether. The combined ether extracts are dried over magnesium sulfate,then evaporated to dryness to yield 23.8 g. (0.164 mole) of the freeebase. This material is dissolved in 70 ml. of diglyme, 3.8 g. (0.164mole) of sodium is added and warmed on a steam bath to effect solution.The solution is cooled to room temperature, 16.3 g. (0.082 mole) of4-methylpiperazine-l-carbonyl chloride is added, and the reactionmixture is stirred at room temperature for one-half hour, then refluxedfor two hours. The insoluble portion is filtered off and washed with 100ml. of diglyme. The filtrate and washings are combined and the diglymeis removed on a steam bath at 20 mm. of Hg. The residue is againfiltered from some solid material and the filtrate is distilled througha Nester-Faust spinning band colum. The fraction boiling at 120-122(0075-010 mm. of Hg) is collected; yield 12.4 g. of Z-diethylaminoethyl4 methylpiperazine-l-carbothiolate, n =1.5110.

The above product is dissolved in 50 ml. of anhydrous ether and treatedwith 300 ml. of anhydrous ether which has previously been saturated withanhydrous hydrogen chloride in an ice-bath. The resulting gum whichseparates is triturated to a solid, filtered and recrystallized fromethanol-ether; yield 12.7 g., melting point 229- 234 C. dec.

Example 1X.Preparation 0f Z-diethylaminoethyl 2,4,5- trimethylpiperazz'lze-I -carb0xylale Metallic sodium (0.4 g.) is dissolved in46.8 g. of 2- diethylaminoethanol followed by the addition of 20.0 g. of1-carbethoxy-2,4,S-trimethylpiperazine. This is treated in a mannersimilar to Example V. The product obtained is2-diethylaminoethyl-2,4,S-trimethylpiperazine-l-carboxylate.

Example X Parenteral solutions of the compounds of the present inventioncan be used as a method of administration. An illustrative formulationis the following.

Percent w./v. Diethylaminoethyl 4-methyl-l-piperazinecarboxylate 25-100Hydrochloric acid as needed to obtain pH, q.s.

to pH 4.5-5.5. Sequestrene, disodium 0.05 Benzyl alcohol reagent 0.9-1.5

Pyrogen-free distilled water to make 100.00 ml.

Example XI The compounds of this invention may be administered in oralsyrup preparations such as the following.

Percent w./v. Diethylaminoethyl 4-methyl-l-piperazinecarboxylate 25-100Sodium phosphate monobasic, sodium phosphate dibasic, q.s. to pH4.5-5.5.

Methylparabens 0.08 Propylparabens 0.02 Sucrose 10-40 Red Dye FDC #20.005 Cherry flavor 0.05

Distilled water, q.s. ad ml.

The above syrup has a pH of about 4.5-5 .5.

Other ingredients may replace those indicated above for example asuspending agent such as bentonite magma, tragacanth,carboxymethylcellulose, methylcellulose, carbopol 934 (carboxy vinylpolymer of high molecular weight), etc. can be used. The phosphates usedabove as buffers can be replaced with citrates or tartrates. Aspreservatives, the parabens can be replaced with alkali metal benzoates,sorbic acid or the like. The sugar and sorbitol could be replaced inWhole or in part with corn syrup, glycerol, other sugars, etc. Andalternate dyes and flavors could replace those indicated above.

Example XII The compounds of this invention may be dispensed in hard orsoft shell capsules. A useful formulation for preparing such capsules isas follows.

Mix thoroughly and dispense in 50 capsules.

Example XIII The com-pounds of this invention may be dispensed aspharmaceutical tablets or Oblets. A useful formulation for preparingtablets or Oblets is as follows.

Per tablet, g. Diethylaminoethyl 4-methyll-piperazinecarboxylate 50.0Corn starch 2.0 Methylcellulose (viscosity 400 cps.) 0.50 Magnesiumstearate, 1% 0.525

Mix thoroughly and dispense as 100 tablets.

.The above tablets each containing 0.5 g. of active ingredient may beconsidered to provide an entire single daily dose. Such a tablet mightbe scored so as to provide fractional daily doses. Alternatively,tablets containing smaller amounts of active ingredient might beprepared for fractional daily or smaller doses.

We claim:

1. A method of treating virus infections in warm blooded animals whichcomprises administering to said animals an antiviral compositioncomprising an edible carrier and from 5 to about 500 mg. per kg. basedon body weight of said animal, of a substituted piperazine of theformula:

wherein R is selected from the group consisting of hydrogen and loweralkyl, R and R are selected from the group consisting of hydrogen andlower alkyl, R and R are lower alkyl, X is lower alkylene, Z is selectedfrom the group consisting of oxygen and sulfur and acid addition andquaternary salts thereof.

2. The method of claim 1 in which the substituted piperazine is2-diethylarninoethyl-4 methylpiperazine-1- carboxylate.

3. The method of claim 1 in which the substituted piperazine is3-diethylaminopropyl-4-rnethy1piperazine-1- carboxylate.

4. A method of claim 1 in which the substituted piperazine isZ-dimethylaminoisopropyl 4-methylpiperazine-1- ca-rb-oxylate.

5. The method of claim 1 in which the substituted piperazine isZ-dimethylaminoethyl 4-methylpiperazinel-carboxylate.

6. The method of claim 1 in which the substituted piperazine is2-diethylaminoethyl piperazine-l-carboxylate.

7. A composition of matter having antiviral activity, in dosage unitform, comprising a pharmaceutical carrier and from about 0.05 to 2 g. ofa substituted piperazine of the formula:

wherein R is selected from the group consisting of hydrogen and loweralkyl, R and R are selected from the group consisting of hydrogen andlower alkyl, R and R are lower alkyl, X is lower alkylene, Z is selectedfrom the group consisting of oxygen and sulfur and acid addition andquaternary salts thereof.

8. A composition of matter in accordance with claim 7 characterized inthat the substituted piperazine is 2- diethyla-minoethyl 4-methylpiperazine-l-carboxylate.

9. A composition of matter in accordance with claim 7 characterized inthat the substituted piperazine is 3- diethylaminopropyl4-methylpiperazine-l-carboxylate.

10. A composition of matter in accordance with claim 7 characterized inthat the substituted piperazine is 2- dimethylaminoisopropyl 4methylpiperazine 1 carboxylate.

11. A composition of matter in accordance with claim 7 characterized inthat the substituted piperazine is 2- dimethylaminoethyl 4methylpiperazine 1 carboxylate.

No references cited.

LEWIS GOTTS, Primary Examiner.

SHEP K. ROSE, Assistant Examiner.

1. A METHOD OF TREATING VIRUS INFECTIONS IN WARM BLOODED ANIMALS WHICHCOMPRISES ADMINISTERING TO SAID ANIMALS AND ANTIVIRAL COMPOSITIONCOMPRISING AN EDIBLE CARRIER AND FROM 5 TO ABOUT 500 MG. PER KG. BASEDON BODY WEIGHT OF SAID ANIMAL, OF A SUBSTITUTED PIPERAZINE OF THEFORMULA: